The rapid rise of melanoma, in the United States, in the absence of effective therapeutic regimens underscores the urgency and importance of obtaining a deeper knowledge about pathogenesis of this disease. In this project we propose that melanoma is driven by subpopulation of CD271 positive tumor initiating cells. Tumor stem cells (TSCs) in other cancers were shown to be resistant to most conventional chemo and radio therapies, to have the ability to self-renew and efficiently replenish the entire tumor cell population eliminated after traditional medical regimens. Thus to design more successful cancer therapy one must isolate and study the properties of respective TSCs. The primary goal of this proposal is to putatively isolate highly enriched melanoma TSCs from broad spectrum of melanomas and to characterize their molecular profile. We will reach this goal by designing and accomplishing following experimental tasks: 1. Identify and prospectively isolate melanoma TSC by CD271 and additional cell surface markers expression using advanced Fluorescent Activated Cell Sorting and in-vivo tumor transplantation assays. 2. Characterize genetic and/or epigenetic alterations that lie at the root of CD271+ MTSC function by performing global gene expression analysis of MTSCs using microarray and lentiviral reporter technologies; confirm critical role of specific gene candidates in MTSC phenotype 3. Analyze MTSC's advanced tumorigenic properties in the environment that closely reflects natural occurrence of this cancer in humans by characterizing invasive characteristics of MTSCs and a functional role of CD271 in human skin reconstruct mouse model.